Method of administering pyruvate and methods of synthesizing pyruvate precursors

ABSTRACT

A method for administering pyruvate is disclosed which comprises administering a therapeutically effective amount of a pyruvate precursor to a mammal in the form of pyruvamide or a pyruvyl-amino acid. The pyruvyl-amino acid is preferably selected from the group comprising pyruvyl-glycine, pyruvyl-alanine, pyruvyl-leucine, pyruvyl-valine, pyruvyl-isoleucine, pyruvyl-phenylalanine, pyruvyl-proline and pyruvyl-sarcosine, and their amides and esters as well as their salts. Associated with the administration of a pyruvate precursor to a mammal in accordance with this invention are improved insulin resistance, lower fasting insulin levels, and reduced fat gain. Novel methods of synthesizing several pyruvate precursors are also disclosed.

TECHNICAL FIELD

present invention relates generally to a method of administeringpyruvate to mammals, and to methods of synthesizing non-salt precursorsto pyruvate.

BACKGROUND ART

Obesity is a multifactorial disease which affects upwards of 25% of theadult population in the United States of America. It is estimated thatin the U.S.A. between 34-50 million adults are obese, with at least 5million of those adults receiving medical treatment for their obesity.The etiology of obesity can range from simple overeating to severehormonal imbalance. However, the great majority of obesity is probablydue to a complex relationship between the many factors that regulateenergy intake and utilization.

Teleologically, obese individuals may be better prepared for survival intime of limited food supply because of their ability to utilize energyin a more efficient manner. However, given that there is almost anunlimited food supply in the U.S.A., this efficiency of energyutilization probably leads to obesity. Furthermore, obesity isassociated with an increased risk of cardiovascular disease, anincreased risk of Type II diabetes, an increased risk of coronary arterydisease, and other chronic diseases. For example, it is believed that inthe U.S.A. there are over 6 million diagnosed cases of obese Type IIdiabetes, with an estimated 4 million cases being undiagnosed.

Given the large obese population and the associated problems, the areaof obesity research and product development for the management ofobesity has been explored, yet the problem remains. U.S. Pat. No.4,351,835 teaches a method for preventing body fat deposition in mammalsby oral administration of a mixture of pyruvate and dihydroxyacetone(DHA).

Subsequent additional research with rats investigated the effect ofpyruvate and DHA under normal dietary conditions. In that study, ratswere fed either a controlled diet or an experimental diet in which partof the carbohydrates were replaced with a 1:1 mixture of pyruvate andDHA, which mixture constituted 15% of the total caloric intake. Ratswhich received the experimental diet gained less weight, and had greaterrates of heat production and energy expenditure than rats receiving acontrol diet. The experimental diet reduced body fat content by 32%without any significant effect on either protein or water content.

Similarly, in another study, Type II diabetic humans were fed 56 gramsof pyruvate and DHA in a 1:1 mixture for seven days, during which timeperiod glucose tolerance and turnover were measured. Reductions infasting blood glucose concentration and peak glucose concentration aftera glucose tolerance test were observed.

Yet another study assessed the relative effectiveness of pyruvate andDHA. In that study, obese Zucker rats were placed in one of four dietgroups. One diet was a control and each of the other diets featured asemi-purified rat diet with only one of the following features: (a) 6%pyruvate, (b) 6% DHA, or (c) 6% pyruvate/DHA (1:1). A number ofphysiologic variables were measured. The conclusion of the study wasthat generally changes due to the addition of DHA or pyruvate/OHA to thediet were not as great as changes due to the addition of only pyruvate.In fact, often the changes due to the addition of DHA or pyruvate/DHAcould either be attributed to feed restriction or to the pyruvate incombination.

Finally, U.S. Pat. No. 4,548,937 discloses a method for minimizingweight gain by adding pyruvate to the diet.

Based on the above studies, the experimental data indicated thatpyruvate was an efficacious compound in altering metabolic variables inrats. Pyruvate, also known as pyruvic acid, is a common metabolite ofthe body.

A problem exists in administering effective dosages of pyruvate tohumans in that heretofore the only ways to supply pyruvate have been inthe form of a liquified pyruvic acid or in the form of the mineral saltsof pyruvate, for example via sodium, potassium or calcium salts. Thesesalts are organoleptically poor, as is tolerance of these salts.Furthermore, in humans the amount of these salts required to obtain theproper dosage of pyruvate for maximal effect raises the electrolytelevel of the recipient to 2-6 times the safe and adequate recommendedlevel when given as a supplement to a typical diet. With respect to theliquid pyruvic acid, the liquid is very acidic and results in the bodyliterally being burned. Attempting to solve the acidity problem throughdilution results in the human body being unable to ingest acceptablelevels of pyruvate.

It is thus apparent that the need exists for an improved method ofadministering pyruvate to humans. It is also apparent that the needexists for an improved method of synthesizing a hydrolyzable precursorfor pyruvate, other than in the form of a pyruvate salt.

DISCLOSURE OF THE INVENTION

There is disclosed a method for administering pyruvate to mammals, whichmethod comprises administering a therapeutically effective amount of apyruvate precursor in the form of pyruvamide or a pyruvyl-amino acid.More preferably the pyruvate precursor constitutes between 2%-20% byweight of the diet of the mammal.

There is also disclosed a method for improving insulin resistance in amammal, as measured by a fasting blood glucose tolerance test, whichmethod comprises administering a therapeutically effective amount of apyruvate precursor in the form of a pyruvyl-amino acid prior toperforming a fasting blood glucose tolerance test, such that it requiresa reduced level of insulin to maintain blood glucose levels in thefasting blood glucose tolerance test than is exhibited in the absence ofthe prior administration of the pyruvate precursor. More preferably thepyruvate precursor constitutes between 2%-20% by weight of the diet ofthe mammal.

There is also disclosed a method for reducing fat deposition in mammals,which method comprises administering a therapeutically effective amountof a pyruvate precursor in the form of a pyruvyl-amino acid.

There are also disclosed methods of synthesizing the pyruvate precursorspyruvamide, pyruvyl-glycine, pyruvyl-alanine, pyruvyl-valine,pyruvyl-leucine, and pyruvyl-isoleucine.

One aspect of the invention provides an effective method foradministering pyruvate to mammals.

Yet another aspect of the invention resides in a relatively easy andcost effective method for synthesizing a pyruvate precursor, other thanin the form of a salt.

Other aspects and advantages of the instant invention will be apparentfrom the following description, examples, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the effect of the ingestion of pyruvate,and pyruvate analogs, using the method of this invention, on daily foodintake.

FIG. 2 is a graph illustrating the effect of the ingestion of pyruvate,and pyruvate analogs, using the method of this invention, on daily bodyweight.

FIG. 3 is a graph illustrating the effect of pyruvate, and pyruvateanalog, on weight gain, body water content and body fat content.

FIG. 4 is a graph illustrating the concentration of pyruvate in the bodyover time after gavage of various pyruvate precursors.

FIGS. 5 and 6 are graphs illustrating the effect of the ingestion ofpyruvate, and pyruvate analogs, on glucose and insulin levels in fattyZucker rats after a glucose tolerance test.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with a dietary supplement which canbe utilized by obese or overweight mammals, as well as mammals havingType II diabetes. The present invention is also concerned with thesynthesis of a precursor for pyruvate, other than in the form of a salt.

Although at this time pyruvate theoretically appears to be the mostefficacious compound in addressing obesity and problems associated withType II diabetes, the utility of pyruvate in humans in the clinicalmanagement of Type I diabetes or obesity has been limited by theelevated mineral load associated with pyruvate salts, which until thistime were the only practical method of supplying pyruvate to the body.Pyruvate can also be supplied as a liquid acid, but it is so acidic thatit must be diluted. When the liquid acid is diluted sufficiently to betolerable, it requires too large a volume of liquid to be consumed inorder to obtain a sufficient ingestion of pyruvate.

Table I illustrates the raised electrolyte levels associated with thegeneration of an effective dose (28 grams) of pyruvate in the form ofpyruvate salts, with the salts being used either alone or incombination.

                  TABLE I    ______________________________________    28 g Pyruvate Na+        K+         Ca++    ______________________________________    Single salt, mg                  7,000      --         --    Single salt, mg                  --         12,560     --    Single salt, mg                  --         --         6,400    Combination*, mg                  3,500      --         3,200    Combination*, mg                  --         6,300      3,200    Combination*, mg                  2,330      4,180      2,140    ESADDI** range, mg                  1100-3300  1875-5625  1,200    ______________________________________     *Each salt is added as an equal proportion of the total 28 grams of     pyruvate.     **Estimated Safe and Adequate Daily Dietary Intake in the RDA 10th     edition.

As can be seen from Table I, the electrolyte level is raised to between2-6 times the level recommended in humans, regardless of how thepyruvate salts are ingested.

In order to dispense or generate pyruvate in mammals, a unique, methodhas been proposed which resulted in a unique synthesis for preparingpyruvamide, a hydrolyzable precursor of pyruvate. Earlier synthesis ofpyruvamide has been by hydrolysis of pyruvonitrile, by the acidhydrolysis of lantacidin A, or by the permanganate oxidation oflactamide.

The unique proposed synthesis of pyruvamide associated with thisinvention utilized the generation of pyruvyl chloride in situ fromsodium pyruvate and thionyl chloride or oxalyl chloride. The pyruvylchloride was then reacted with 1,1,1,3,3,3-hexamethyldisilazane (HMDS)at a temperature of 0°-40° Centigrade, followed by treatment withmethanol in the presence of a suitable solvent such as dichloromethane(DCM), tetrahydrofuran (THF), or dimethylformamide (DMF) to generate thepyruvamide. The yield of pyruvamide was in the range of 50-80%. It wasthen discovered that higher yields and less colored by-products wereformed by replacing the thionyl chloride with oxalyl chloride. Thisimproved the method of obtaining the pyruvyl chloride intermediate.

The pyruvate analog, pyruvamide, and its unique synthesis were believedto have solved the problem as to a usable pyruvate source, since thisparticular synthesis did not require ultimate ingestion of a pyruvatemineral salt. Rather surprisingly, in subsequent studies with rats, itwas found that the rats apparently did not exhibit a tolerance forpyruvamide at the dosage used. Rats in that particular test groupconsumed insignificant quantities of food, and while the rats lostappreciable body weight, this was believed to be due to the consumptionof an insignificant quantity of food and not due to any desirablereasons. The reason for the rats' surprising aversion to the dietcomprising pyruvamide was not completely understood, and requiredfurther study and testing.

Since it was initially determined that pyruvamide is not a preferredpyruvate analog, other pyruvate precursors were investigated. It wasthen found that other hydrolyzable, non-salt, pyruvate precursors couldbe synthesized. For example, an improved synthesis of pyruvyl-glycinevia a benzyl ester protection method, was discovered. The traditionalmethod of synthesis of pyruvyl-glycine involves reacting phosphorousoxychloride or p-toluene sulfonyl chloride with pyravic acid to yieldpyruvyl chloride. The pyruvyl chloride formed using the traditionalsynthesis is then reacted with glycine benzyl ester hydrochloride toform pyruvyl-glycine benzyl ester, or with glycine in pyridine to yieldpyruvyl-glycine. The yield of pyruvyl-glycine benzyl ester was about35%, and the yield of pyruvyl-glycine was about 12%.

The improved method of synthesis involves the reaction of sodiumpyruvate with thionyl chloride or preferably with oxalyl chloride toform pyruvyl chloride. The pyruvyl chloride is then reacted with glycinebenzyl ester hydrochloride to form the pyruvyl-glycine benzyl ester,with a yield of about 85%. The pyruvyl-glycine benzyl ester thenundergoes hydrogenolysis to obtain pyruvyl-glycine with a yield of about90%. It can be readily appreciated that the ultimate yield ofpyruvyl-glycine using this improved method is substantially higher thanthe yield associated with the traditional method of synthesis. Thisimproved method is set forth below.

Preferably in this method of synthesis, the thionyl chloride or oxalylchloride is added to a suspension of sodium pyruvate to yield thepyruvyl chloride to which is added the of glycine benzyl esterhydrochloride. The resulting slurry is cooled during the addition ofN-methylmorpholine solution to yield a pyruvyl-glycine benzyl ester. Theyielding of the pyruvyl-glycine benzyl ester occurs after warming, theaddition of water, separation, washing, drying, evaporation,precipitation, filtering, and drying again. The pyruvyl-glycine benzylester is then charged in a hydrogen pressure vessel in a solvent and apalladium on carbon catalyst, with the hydrogen pressure beingapproximately 60 psi such that hydrogen is digested whereby followingfiltering, concentration, precipitation, filtering, and drying there isyielded pyruvyl-glycine.

More preferably the thionyl chloride or oxalyl chloride is added to amechanically stirred suspension of sodium pyruvate in dichloromethanewhereby a gas is evolved and pyruvyl chloride is yielded. The pyruvylchloride is cooled during the dropwise addition of glycine benzyl estersolution to yield pyruvyl-glycine benzyl ester following the warming toroom temperature, the addition of water, separation, washing withdiluted hydrochloric acid and brine, drying with magnesium sulfate,evaporation of the dichloromethane, precipitation, filtering and dryingagain. The pyruvyl-glycine benzyl ester is then charged in a hydrogenpressure vessel with ethyl acetate and 4% to 20% palladium on carbon,with the hydrogen pressure being approximately 40-150 psi, such thathydrogen is digested whereby following warming, filtering,concentration, precipitation, filtering and drying there is yieldedpyruvyl-glycine.

For example to synthesize the pyruvyl-glycine benzyl ester set forthabove, a stirred suspension of glycine benzyl ester hydrochloride (182g, 0.9 mol) in dichloromethane (500 ml) is added drop wise at roomtemperature to N-methlymorpholine (220 ml, 2 mol). After approximately 2hours, the solution is refrigerated and the precipitated salt isfiltered and discarded prior to use. Additionally, to a mechanicallystirred suspension of sodium pyruvate (110 g, 1 mol) in dichloromethane(1 L) is added oxalyl chloride (110 ml, 1 mol) in one portion. Theevolution of gas subsides after approximately 5 hours. The pale yellowslurry is kept under a blanket of nitrogen and cooled to between -20°and -30° Centigrade during the dropwise addition of the glycine benzylester hydrochloride solution prepared above. The slurry is then warmedto room temperature. Water (1 L) is added. The lower organic layer isseparated and washed with diluted HCl (3 times with 500 ml), brine (2times with 250 ml), and finally dried with magnesium sulfate. Thedichloromethane is then evaporated and finally when precipitationbegins, heptane is slowly added to increase productivity. The sandysolid is filtered and dried to a constant weight.

A hydrogen pressure vessel is charged with the pyruvyl-glycine benzylester (120 g, 0.51 mol), ethyl acetate (480 ml), and 4% palladium oncarbon (12 g, 10% by weight). Hydrogen pressure is adjusted toapproximately 60 psi, the temperature to approximately 40° Centigradeand the reagents stirred until a stoichiometric amount of hydrogen isdigested. The solution is then filtered through 1.5 micron glass fiberfilter and concentrated. When product starts precipitating, heptane isagain added to increase productivity. The precipitate is filtered anddried to a white solid of constant weight which is pyruvyl-glycine.

A method of synthesizing pyruvyl-alanine or pyruvyl-glycine according tothe invention comprises the steps of reacting sodium pyruvate withoxalyl chloride to yield pyruvyl chloride, then reacting pyruvylchloride with a suspension of alanine trimethylsilyl ester hydrochlorideto yield a pyruvyl-alanine trimethylsilyl ester and then subjecting thepyruvyl-alanine trimethylsilyl ester to hydrolysis to yieldpyruvyl-alanine. The pyruvyl-alanine synthesized via the method setforth above serves as a pyruvyl-amino acid pyruvate precursor.

A method of synthesizing pyruvyl-valine according to the inventioncomprises the steps of reacting sodium pyruvate with oxalyl chloride toyield pyruvyl chloride, then reacting the pyruvyl chloride with asuspension of valine trimethylsilyl ester hydrochloride to yieldpyruvyl-valine trimethylsilyl ester and then subjecting thepyruvyl-valine trimethylsilyl ester to hydrolysis to yieldpyruvyl-valine. The pyruvyl-valine synthesized via the route set forthabove serves as a pyruvyl-amino acid pyruvate precursor.

A method of synthesizing pyruvyl-leucine according to the inventioncomprises the steps of reacting sodium pyruvate with oxalyl chloride toyield pyruvyl chloride, then reacting the pyruvyl chloride with asuspension of leucine trimethylsilyl ester hydrochloride to yield apyruvyl-leucine trimethylsilyl ester and then subjecting the pyruvylleucine trimethylsilyl ester to hydrolysis to yield pyruvyl-leucine. Thepyruvyl-leucine synthesized via the route set forth above serves as apyruvyl-amino acid pyruvate precursor.

A method of synthesizing pyruvyl-isoleucine according to the inventionthe steps of reacting sodium pyruvate with oxalyl chloride to yieldpyruvyl chloride, then reacting the pyruvyl chloride with a suspensionof isoleucine trimethylsilyl ester hydrochloride to yieldpyruvyl-isoleucine trimethylsilyl ester and then subjecting thepyruvyl-isoleucine trimethylsilyl ester to hydrolysis to yieldpyruvyl-isoleucine. The pyruvyl-isoleucine synthesized via the methodset forth above serves as a pyruvyl-amino acid pyruvate precursor.

A method of synthesizing pyruvyl-phenylalanine according to theinvention comprises the steps of reacting sodium pyruvate with oxalylchloride to yield pyruvyl chloride, then reacting pyruvyl chloride witha suspension of phenylalanine trimethylsilyl ester hydrochloride toyield pyruvyl-phenylalanine trimethylsilyl ester and then subjecting thepyruvyl-phenylalanine trimethylsilyl ester to hydrolysis to yieldpyruvyl-phenylalanine. The pyruvyl-phenylalanine synthesized via theroute set forth above serves as a pyruvyl-amino acid pyruvate precursor.

A method of synthesizing the pyruvate precursor pyruvamide, inaccordance with the invention comprises the steps of preparing pyruvylchloride by reacting sodium pyruvate with thionyl chloride or oxalylchloride to yield pyruvyl chloride, and reacting the pyruvyl chloridewith 1,1,1,3,3,3-hexamethyldisilazane at a temperature of 0°-40°Centigrade, followed by treatment with methanol in the presence of asolvent to generate pyruvamide. Preferably the solvent is selected fromthe group consisting of, but not limited to, dichloromethane,acetonitrile, tetrahydrofuran and dimethylformamide.

Other pyruvate analogs were synthesized, for example with the improvedmethod of synthesis having oxalyl chloride substituted for phosphorousoxychloride or thionyl chloride. The other steps in the syntheses of theother analogs correspond to the steps in the synthesis of thepyruvyl-glycine. While pyruvyl-amino acid precursors comprising alanine,valine, leucine, isoleucine, and phenylalanine could be synthesizedusing the benzyl ester hydrochloride of the particular amino acid, itwas discovered that a preferable synthesis involved their substitutionby the trimethylsilyl esters of the respective amino acid.

The most preferred method of synthesizing pyruvyl-glycine will now bedescribed. This method employees inexpensive starting materials, is aone-step synthesis, and has a productivity level that is similar to orsuperior to the other methods disclosed herein.

In an example of a first embodiment of this most preferred method ofsynthesizing pyruvyl-glycine a 2 L flask (stirrer, condenser, nitrogen)was charged with glycine (15 g, 0.2 mol), anhydrous tetrahydrofuran(400mL) and trimethylsilyl chloride (22 g, 0.2 mol). The reactants werestirred at reflux for 4 hours. The solution was then cooled to 0° C. ina cooling bath. Separately, to sodium pyruvate (22 g, 0.2 mol) inmethylene chloride (400 mL) at 0° C. was added dropwise a solution ofoxalyl chloride (26 g, 0.2 mol) in methylene chloride (40 mL). Theslurry was then stirred at 25° C. for 2 hours under a blanket ofnitrogen. The precipitated sodium chloride was filtered off and thefiltrate was added to the above solution of glycine trimethylsilyl esterat 0° C. The condenser was replaced by an addition funnel and a solutionof propylene oxide (23 g, 0.4 mol) in methylene chloride (50 mL) wasadded dropwise at 0° C. The reaction mixture was then stirred to 25° C.over a 2 hour period until clear. Methanol (80 mL) was added at once andthe solvents were evaporated. The solid residue (36 g) wasrecrystallized from ethyl acetate/heptane to a tan solid, mp 86°-7° C.(17 g, 59%).

In an example of a second embodiment of this most preferred method ofsynthesizing pyruvyl-glycine a 2 L flash (stirrer, condenser, nitrogen)was charged with glycine (15 g, 0.2 mol), anhydrous dioxane (400 mL) andtrimethylsilyl chloride (22 g, 0.2 mol). The reactants are stirred atreflux for 4 hours. The solution is then cooled to 0° C. in an ice bath.In a separate flask, oxalyl chloride (26 g, 0.2 mol) is added to sodiumpyruvate (22 g, 0.2 mol) in ethyl acetate (400 mL) at 0° C. The slurrywas stirred at 25° C. for 2 hours under a blanket of nitrogen. Theprecipitated sodium chloride was filtered off and the filtrate was addedto the above solution of glycine trimethylsilyl ester at 0° C. Thecondenser was replaced by an addition funnel and a solution of propyleneoxide (23 g, 0.4 mol) in ethyl acetate (50 mL) was added dropwise at 0°C. The reaction mixture was then stirred at 40° C. for 2 hours andovernight at 20° C. Methanol (80 mL) was added at once and the solventswere evaporated. The solid residue (34 g) was recrystallized from ethylacetate/heptane to a tan solid, mp 87° C. (22 g, 76%).

The most preferred one-step synthesis of pyruvyl-glycine comprisescharging a 2 L flask (air stirrer, condenser, nitrogen inlet) withglycine (15 g, 0.2 mol), anhydrous acetonitrile (ACN)(500 mL) andtrimethylsilyl chloride (30.5 mL, 0.24 mol). The reactants are stirredat reflux for 3 hours. The clear turbid solution is then cooled to 5° C.in an ice bath and propylene oxide (100 mL) is added at once. In aseparate flask, oxalyl chloride (21 mL, 0.24 mol) is added to sodiumpyruvate (24.2 g, 0.22 mol) in ACN (500 mL) at 0° C. with vigorousdegassing. The solution is stirred at 25° C. for 3 hours under a blanketof nitrogen. The slurry is cooled to 5° C. and added in one portion tothe above trimethylsilyl ester solution at 5° C. The sodium chloride isnot filtered off at this stage. The slurry is stirred at 5° C. for about2 hours and then warmed to 25° C. and held overnight. Methanol (80 mL)is added at once, the salts are filtered off and the solvents areevaporated at a temperature of less than 35° C. The residue (about 74 g)is flash-filtered through a bed of silica (200 g) with ethylacetate/heptane (1:1). The fractions containing the product areconcentrated to a solid mass (26 g, 90%). Following recrystallizationfrom chloroform/heptane (1:3 150 mL), a tan solid is obtained, meltingpoint 87° C. (21.9 g, 75%). The pyruvyl-glycine is recrystallized usingany suitable procedure such as freeze drying, spray drying or microwavevacuum drying. This one-step synthesis presents several advantages: (a)the scavenging of the acid liberated during the pyruvylation of theamino acid derivative is only slightly exothermic, thus giving bettercontrol over the reaction and necessitating little cooling during thereaction,; (b) the by-product (i.e. chloropropanols) can easily beremoved by evaporation, thus avoiding the aqueous elimination of thesalts generated by other methods; and (c) the cost of propylene oxide isnegligible as compared to traditional organic bases.

The invention will be better understood in view of the followingexamples which are illustrative only and should not be construed aslimiting the claims of invention.

EXAMPLE 1 Comparison Testing

The following study was designed to determine the efficacy of onepyruvate analog, pyruvyl-glycine, administered as a dietary supplementto rats.

Experimental groups were formed having 8 rats in each group. For oneweek prior to the beginning of the test period, the rats were housed inindividual cages and provided a powdered controlled diet to acclimatethe rats to their surroundings. Familiarization of the rats with thepowdered diet was determined by consistent weight gain and feed intakefor no less than four days. Group 1 consisted of rats receiving a dietof 26% protein, 12.1% fat, and 62% carbohydrate (total kcals) pair-fedaccording to the experimental group that consumed the least amount offood. Each of the diets except that fed to the control group featured adiet comprised of 6% pyruvate (by calories). Experimental group 1consisted of rats that received the controlled diet with the 9% calciumpyruvate substituted for sucrose (by weight). Experimental group 2consisted of rats that received the controlled diet with 7.2% pyruvamidesubstituted for sucrose (by weight). Experimental group 3 consisted ofrats that received a controlled diet with 12.2% pyruvyl-glycinesubstituted for sucrose (by weight).

Food bowls for all 4 groups were replenished with the diets on a dailybasis. Food consumption for the previous day and body weights of therats were recorded, and the animals were pair-fed to the group consumingthe least amount. The rats were maintained on their respective diets foreight days. Set forth below in Table II are the actual ingredientsassociated with each of the four test groups.

                  TABLE II    ______________________________________                       Calcium            Pyruvyl-    DIET     Control   Pyruvate Pyruvamide                                          glycine    ______________________________________    Ingredient, g    Casein   200       200      200       200    Methionine             3         3        3         3    Starch   250       250      250       250    Sucrose  358       310      286       236    Cellulose             50        50       50        50    Corn Oil 50        50       50        50    Vitamins 10        10       10        10    Choline  2         2        2         2    Salt Mix 5         5        5         5    Ca/PO4   72        30       72        72    CaPyruvate             0         90       0         0    PyrAmide 0         0        72        0    PyrGlycine             0         0        0         122    TOTAL    1000      1000     1000      1000    ______________________________________

As can be seen in FIG. 1, the food intake for the control group, thepyruvate group and the pyruvyl-glycine group all were somewhat similarespecially through the time period of days 4-7. However, as has beendiscussed above, and as can be seen in FIG. 1, the daily food intake ofthe dietary treatment comprising pyruvamide was extremely insignificant.

As can be seen in FIG. 2, the body weight in grams of the control groupincreased from approximately 205 to approximately 230. Meanwhile, thepyruvate fed group exhibited a statistically significant smallerincrease in body weight. However, as has been pointed out above, the useof the mineral salts of pyruvate in humans is precluded due to the highelectrolyte levels produced. As can be seen, the pyruvyl-glycine fedtest group also exhibited a lesser increase in body weight as comparedto the control group. Finally, the pyruvamide test group displayed asharp decrease in body weight. However, that was due to theinsignificant amount of food consumed by these rats due to what wasinitially believed to have been a low tolerance to pyruvamide at theselevels.

Based on these short duration test results, pyruvyl-glycine was found tohave excellent potential as a substitute for pyruvate, becausepyruvyl-glycine exhibited biological activity, exhibited no noticeabletoxicity, and it eliminated the excessive mineral load associated withpyruvate salts while retaining chemical stability. In previous studies,pyruvyl-glycine has been shown to be as stable as the mineral salt ofpyruvate in casein diets (less than 3% loss at 37° Centigrade over 5days), while pyruvamide appeared more unstable (73% loss under similarconditions). Additionally, pyruvyl-glycine is also stable in simulatedsterilization conditions (less than 5% loss at 125° Centigrade over 8minutes) while the pyruvamide loss is higher (greater than 50% lossunder similar conditions).

EXAMPLE 2 Comparison Testing

The longer term effects of pyruvate and pyruvyl-glycine on feed intakeand weight gain of the rats in the comparison test were investigated byextending the testing for the control group and the two groups with thepyruvate dietary supplement and the pyruvyl-glycine dietary supplement.

Beginning after the eighth day of the test, the three groups were eachfed at a reduced level of 15 g per day. The results of this test areshown in Table III.

                  TABLE III    ______________________________________                Control                       Pyruvate Pyruvyl-Glycine                Group  Group    Group    ______________________________________    Food Consumption, g    Day 0-8       152.1    132.2    111.6    Day 8-23      225      225      225    Day 0-23      377.1    357.2    336.6    Avg daily intake, g                  16.4     15.5     14.6    Weight Gain, g    Day 0-8       27.8     13.7     12.4    Day 8-23      52.3     50.2     45.5    Day 0-23      80       64       57.8    Avg daily gain, g                  3.48     2.78     2.51    Initial wt, g 205.4    205.6    202.3    Final wt, g   285.5    269.6    260.1    ______________________________________

From day 8 to 23, weight gain was much more similar in each of thegroups although the least in the pyruvyl-glycine group. Consumption ofthe pyruvyl-glycine dietary treatment resulted in a 12% lower carcassweight, a 50% decrease in retroperitoneal fat pad weight, and moststrikingly, a lower percentage of total body fat, as shown in FIG. 3. Infact, in only 3 weeks, the group fed the dietary treatment comprisingpyruvyl-glycine exhibited a 30% decrease in body fat content whencompared to the other experimental groups.

Previous experiments have shown that the addition of pyruvate orpyruvate with DHA to the diet of rats causes a reduction in expectedbody weight gain. In this experiment, the addition of pyruvate to thediet had a suppressive effect on weight gain although no effect oncarcass composition. Although pyruvate did not have a measurable effecton carcass composition, pyruvyl-glycine had a dramatic effect onreducing the body fat content. This difference is remarkable given therelatively short length of the feeding trial. Despite the dramaticchanges in fat content, lean body mass (protein) and ash levels were notadversely affected. In fact, as a percentage of body weight, protein andash content were elevated in the pyruvyl-glycine fed group of rats.

The possible reasons for the enhanced effect of pyruvyl-glycine are notclearly understood. It is theorized that perhaps the enhanced effectcould be the result of: (1) the addition glycine having a synergisticenhancement of the pyruvate effect; (2) the pyruvyl-glycine having ametabolic effect independent of either glycine or pyruvate; and/or (3)the pyruvate being stabilized by the linkage with glycine, such that thepyruvate is delivered to the target tissues at higher levels.

Additional tests were conducted to evaluate the actual effect ofingestion of non-salt pyruvate analogs on the pyruvate level in blood.FIG. 4 shows the level of pyruvate concentration in millimoles per literof blood over a period of time for rats fed pyruvate, a pyruvate/glycinemixture, pyruvyl-glycine, pyruvyl-alanine, pyruvyl-leucine, andpyruvamide. It was expected that these tests would confirm the efficacyof pyruvyl-glycine as a pyruvate precursor.

As can be seen in FIG. 4, the administration of pyruvate caused theconcentration of pyruvate in the blood to increase. Similarly, and forreasons still not fully understood, the oral ingestion of pyruvate withglycine resulted in a higher concentration of pyruvate ultimatelyevidencing itself in the blood. It was somewhat surprising therefore tosee the change in the pyruvate level when pyruvyl-glycine was ingestedand broke down to form pyruvate and glycine which could then bemeasured. Thus, it was found that pyruvyl-glycine caused a slightincrease in pyruvate and a substantial increase in glycine, therefore itwas an acceptable pyruvate analog, although it was perhaps not the bestanalog available. Subsequent tests with pyruvyl-alanine andpyruvyl-leucine showed that these two pyruvyl-amino acids were in factcapable of supplying greater amounts of pyruvate to the body over alesser amount of time.

The level of pyruvate concentration in the blood followingadministration of pyruvamide was then tested. Surprisingly the pyruvateconcentration experienced its most dramatic increases. As can be seen inFIG. 4, the pyruvate concentration after 1 hour was almost four times ashigh when compared with ingestion of the other analogs. Based on thisdevelopment it was theorized that the apparent intolerance to pyruvamidein the earlier tests was caused not by an aversion to any ingestion ofpyruvamide, but because the pyruvamide was converted far more quickly topyruvate, therefore requiring far less total ingestion of the analog toobtain the desired result. Thus, a decrease in the amount of pyruvamideactually allowed to be ingested is now believed to provide the bestsolution to the problem.

EXAMPLE 3 Comparison Testing

The following study was designed to determine the efficacy of onepyruvate analog, pyruvyl-glycine, administered to rats having problemswith obesity and Type II diabetes (fatty Zucker rat). These rats werefed for 21 days diets identical as to those described in Example 1.These rats experience difficulty in controlling blood glucose levels sothey must produce high amounts of insulin in order to keep blood glucosenormal.

After 21 days of feeding the respective diets, the pyruvate-fed and thepyruvyl-glycine-fed rats had reduced fasting insulin levels, yet fastingblood glucose was not negatively affected. This is shown in FIGS. 5 and6. A glucose tolerance test was then administered to these rats bygavaging 1 g glucose/kg body weight. The blood glucose was similar inall treatment groups but the insulin levels in the pyruvate fed group,and especially in the pyruvyl-glycine fed group, were lower than in thecontrol fed group. This indicates that the rats fed pyruvate andpyruval-glycine result in reduced insulin resistance in these fattyZucker rats.

The dietary supplement of this invention could be utilized in a completenutritional, similar to ENSURE® which is a nutritional productdistributed by Ross Laboratories, Columbus, Ohio, as a dietarysupplement which could be added to a drink, or as a dietary supplementconsumable in tablet form, as individual preference determines. Othermethods of administration could be via enteral, parenteral, or otheroral delivery systems. Similarly, the preferable level of usage would beanywhere from 0.5% to 20% by weight of the diet, dependent upon theresults desired.

While the method for administering pyruvate and more particularly themethods of synthesizing non-salt precursors to pyruvate herein describedconstitute preferred embodiments of this invention, it is to beunderstood that the invention is not limited to this precise form ofmethod and that changes may be made therein without departing from thescope of the invention which is defined in the appended claims.

What is claimed is:
 1. A method for administering pyruvate to mammals,which method comprises orally ingesting a therapeutically effectiveamount of a pyruvate precursor, said precursor being in the form of acovalently linked pyruvyl-amino acid compound.
 2. A method foradministering pyruvate to mammals according to claim 1, wherein saidorally ingested covalently linked pyruvate precursor compound isselected from the group comprising pyruvyl-glycine, pyruvyl-alanine,pyruvyl-leucine, pyruvyl-valine, pyruvyl-isoleucine,pyruvyl-phenylalanine, and mixtures thereof.
 3. A method foradministering pyruvate to mammals according to claim 1 wherein theorally ingested pyruvate precursor constitutes between 0.5%-20% byweight of the mammals diet.
 4. A method for administering pyruvate tomammals according to claim 1, wherein said orally ingested covalentlylinked pyruvate precursor compound is pyruvyl-glycine.
 5. A method forreducing body fat deposition in mammals, which method comprises orallyingesting a therapeutically effective amount of a pyruvate precursor,said precursor being in the form of a covalently linked pyruvyl-aminoacid compound, said oral ingestion of said pyruvyl-amino acid compoundcausing a reduction in body fat deposition and a reduction of bodyweight increase.
 6. A method for reducing body fat deposition in mammalsaccording to claim 8 wherein the orally ingested covalently linkedpyruvate precursor compound is selected from the group comprisingpyruvyl-glycine, pyruvyl-alanine, pyruvyl-leucine, pyruvyl-valine,pyruvyl-isoleucine, pyruvyl-phenylalanine, and mixtures thereof.
 7. Amethod for reducing body fat deposition in mammals according to claim 9wherein the orally ingested covalently linked pyruvate precursorcompound is pyruvyl-glycine.
 8. A method for reducing body fatdeposition in mammals according to claim 8 wherein the orally ingestedpyruvate precursor constitutes between 0.5%-20% by weight of themammal's diet.